Plugin error from diversity: Metadata error for alpha group significance

Hi everyone, I am a new user here and here in my first run. Whenever I try the qiime diversity alpha-group significance, I get the error attached below.


I have also attached a screenshot of my metadata. Please let me know where I am going wrong. I am processing 16S sequences from IonTorrent for microbiome analysis

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Hi Sai!
It looks like your problem is that you do not have any categorical metadata in your metadata. For alpha-group significance to work, it requires at least one variable to run statistics with. For example, in the moving pictures tutorial, the metadata contains variables such as “body-site” and “subject”. So, to resolve this you simply need to add a new column to your metadata file with actual information about your samples (as opposed to just sequencing quality information). Ideally, it would be data that helps you answer your research questions, otherwise you may not need investigate alpha group significance at all. I hope that helps!


Thank you so much for your reply. I added some categorical data and still got the same error attached here.

My metadata file is also attached here.

Thanks a million!

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Hi! You need to have categorical data that divide your samples on two or several groups, between which you want to perform analysis to see if there any differences in diversity. It can be different treatments, times of collection of the samples and any other information against which you want to run your analysis.
In your metadata all samples have the same information that causing the error.
Also you have a ‘Day’ column, but it’s a numeric one, you can redo it or add another one with values format like day_0, day_35 in order it to be categorical

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Hello Aeriel,

Thank you very much for your helpful reply. I have a question related to this categorical variable in metadata file. I wanted to ask when do we have categorical variable in the metadata and when we don’t have it? (If I understood correctly, the output of some analyses give us the metadata table with a categorical variable column and in some not! Why is that?)
I don’t know if my question was clear or not :slight_smile:


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Thank you so much! I tried your suggestion and it works now!

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Hey Armin,

There are a couple things I can address in your question. First, the metadata file is not the output of another analysis. This is extra data you should be collecting during your experiment. I usually build mine in excel, but they need to be saved as a .txt file to use in QIIME.

Categorical data is a variable that groups the data, but it isn’t something you can count. For example, in the metadata file Sai attached, there is a column called Body-site and a column called Day. The way it is expressed, day is numeric because you can count, and body site is categorical.

Depending on your research question, you may or may not have categorical data. If you only have numeric metadata, you could consider alpha-correlation instead of alpha-group significance.

Let me know if that doesn’t answer your question :slight_smile:


Hey Aeriel,

Thank you so very much for your wonderful answer and exploitation. It was wonderful and I have learnt a lot.

Thanks again,


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